Structural and Molecular Mechanisms of Cytokine-Mediated Endocrine Resistance in Human Breast Cancer Cells.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKß establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKß signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.

Dual-mechanism estrogen receptor inhibitors.

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Chemical systems biology reveals mechanisms of glucocorticoid receptor signaling.

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, ligand class analysis, to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle-sparing or anabolic for protein balance and mitochondrial potential. Ligand class analysis defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.

Activation of Crtc2/Creb1 in skeletal muscle enhances weight loss during intermittent fasting.

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a major clinical problem for the treatment of estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor, and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.

Predictive features of ligand-specific signaling through the estrogen receptor.

Some estrogen receptor-α (ERα)-targeted breast cancer therapies such as tamoxifen have tissue-selective or cell-specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell-specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X-ray crystallography. Ligands that regulate the dynamics and stability of the coactivator-binding site in the C-terminal ligand-binding domain, called activation function-2 (AF-2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter-atomic distance in the ligand-binding domain predicted their proliferative effects. In contrast, the N-terminal coactivator-binding site, activation function-1 (AF-1), determined cell-specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.

Synthesis of novel steroidal agonists, partial agonists, and antagonists for the glucocorticoid receptor.

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.

Endophthalmitis after penetrating keratoplasty.

PURPOSE: To determine the incidence of endophthalmitis after penetrating keratoplasty (PK) and patient and donor risk factors. DESIGN: Retrospective cohort study using national transplant registry data. PARTICIPANTS: All corneal transplant recipients (n = 11 320) registered on the United Kingdom Transplant Registry undergoing their first PK between April 1999 and December 2006. METHODS: Patients who developed endophthalmitis were identified on the transplant registry. In addition, cases where the fellow cornea from the same donor had been transplanted were included. Clinical information regarding donor and recipient characteristics, surgical details, and postoperative outcomes were collected and analyzed. In cases where endophthalmitis was reported, the diagnosis was verified by a follow-up supplementary questionnaire to the surgeon. Logistic regression was used to investigate differences in the factors associated with the development of endophthalmitis. MAIN OUTCOME MEASURES: Incidence of endophthalmitis and graft survival. RESULTS: The overall incidence of endophthalmitis occurring after primary PK in the UK was 0.67%. The incidence of endophthalmitis occurring within 6 weeks of surgery was 0.16%. Graft survival after endophthalmitis was 27% (95% confidence interval, 16-38) at 5 years, with a mean best-corrected visual acuity of 1.13 (logarithm of the minimum angle of resolution) for surviving grafts. Factors associated with endophthalmitis were donor cause of death (infection), high-risk cases, and indication for corneal transplantation. CONCLUSION: Endophthalmitis remains a serious issue, with those affected having reduced graft survival and poor visual outcomes. Management of the identified recipient and donor risk factors are important to reduce endophthalmitis risk. In particular, the increased incidence of endophthalmitis when the donor dies of infection requires further explanation and review of current donor eye retrieval and eye bank practices. The delayed presentation of endophthalmitis cases also raises questions regarding possible sequestration of microbes within the corneal tissue and the effect of antimicrobials in storage media.

Ligand-binding dynamics rewire cellular signaling via estrogen receptor-α.

Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands showed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways and reveals a new role of the DBD in allosteric control of ERα-mediated signaling.

Changes in ocular monochromatic higher-order aberrations in the aging eye.

PURPOSE: To characterize corneal, internal, and total ocular monochromatic higher-order aberration (MHOA) changes that occur in the aging eye. METHODS: Prospective observational case series including 300 eyes of 167 patients (mean age = 63.8 years) attending the ophthalmology service at University Hospital Ayr, Scotland. Corneal, internal, and total ocular aberrations were measured over a 6-mm dilated pupil. Zernike coefficients were obtained to the sixth order. Changes in MHOA between age groups and inter-eye correlations between right and left eyes were analyzed. RESULTS: A significant inter-eye correlation was found for refractive mean spherical equivalent and cylinder. A significant inter-eye correlation for the whole eye, corneal, and internal MHOA was found (p < 0.001). Right eye analysis found a significant positive correlation between age and the root mean square of whole eye MHOA (p = 0.012), with an increase from 0.517 µm in the fifth decade to 0.824 µm in the ninth. Total internal MHOA increased from 0.411 to 0.704 µm. A significant positive correlation was found between age and internal fourth- (p = 0.007), fifth- (p = 0.029), and sixth-order (p = 0.025) root mean square aberrations. There were no significant age-related changes in corneal MHOA or corneal spherical aberration. Overall mean (SD) corneal SA was 0.203 (0.082) µm. CONCLUSIONS: A strong correlation between the right and left eyes exists for MHOA. Whole eye MHOA increases with age. Such changes can be attributed to age-related changes in the internal optical quality of the eye. Such normative data are useful to the cataract surgeon when considering the use of an aspherical IOL to counteract corneal-induced SA during cataract surgery.

Evolution of operating microscopes and development of 3D visualization systems for intraocular surgery.

The recent development of high-resolution, heads-up, 3D visualization microscopy systems has provided new technical and visualization options for ophthalmic surgeons. In this review, we explore the evolution of microscope technologies, the science behind modern 3D visualization microscopy systems, and the practical benefits (as well as disadvantages) that these systems provide over conventional microscopes for intraocular surgical practice. Overall, modern 3D visualization systems reduce the requirements for artificial illumination and provide enhanced visualization and resolution of ocular structures, improving ergonomics, and facilitating a superior educational experience. Even when considering their disadvantages, such as those related to technical feasibility, 3D visualization systems have an overall positive benefit/risk ratio. It is hoped these systems will be adopted into routine clinical practice, pending further clinical evidence on the benefits they may provide on clinical outcomes.

One-year follow-up of a multifocal intraocular lens with optimized elevated phase shift.

PURPOSE: To evaluate the standard outcomes of a multifocal intraocular lens (mIOL) with optimized elevated phase shift (EPS). SETTING: Qvision, Ophthalmology Department, VITHAS Almería, Spain. DESIGN: Retrospective observational. METHODS: 41 patients, consecutively operated on cataracts or refractive lens exchange with the implantation of the Liberty 640PM (EPS 2.0) and followed during 12 months, were included in the analysis. Retrieved variables were visual acuities at far, intermediate, and near distances; defocus curves (VADC); and prediction error of 4 formulas optimized for IOLMaster 500 and Pentacam AXL Wave. Patient-reported outcomes were also obtained for assessing spectacle independence, satisfaction, bothersome to dysphotopsia, difficulties in daily life tasks, and decision to be operated with the same mIOL. RESULTS: The median monocular efficacy with best distance correction was 0, 0.1, and 0.1 logMAR at far, intermediate, and near distances, respectively, with patients achieving binocularly a median of 0 logMAR at the 3 distances. VADC showed a depth of field of 3 diopters (D) above 0.2 logMAR with a median increase of 0.07 logMAR from -1.5 to -2.5 D. Complete spectacle independence was achieved at far distance, whereas 97.6% and 85.4% was achieved at intermediate and near distances, respectively. 7.3% of patients were bothered by dysphotopsia, and 92.6% of patients were likely to be operated again. CONCLUSIONS: EPS 2.0 restored patients' vision in the full range of the depth of field with a nearly monotone decrease of visual performance from far to near, achieving high rates of spectacle independence at all distances and with low positive dysphotopsia rates ( ClinicalTrials.gov Identifier: NCT05735990).

Approach to a spontaneously ruptured posterior polar cataract.

A 61-year-old man presented with gradual blurring of vision and glare in both eyes for a couple of years, with worsening of the vision in his right eye over the past 2 months. He had no medical history of note. On clinical examination, his visual acuities were 20/80 in the right eye and 20/30 in the left eye, uncorrected. The cornea was clear, and the anterior chamber (AC) was deep in both eyes. He had bilateral mild nuclear sclerosis with round central onion-ring-like posterior opacities. The opacities measured approximately 2 mm in diameter and were marginally larger in the right eye than in the left eye (Figure 1JOURNAL/jcrs/04.03/02158034-202305000-00018/figure1/v/2023-04-20T184543Z/r/image-tiff). In addition, there were 2 curvilinear lines across the right posterior capsule (PC), one of which passed across the lens opacity (Figure 2JOURNAL/jcrs/04.03/02158034-202305000-00018/figure2/v/2023-04-20T184543Z/r/image-tiff). Fundus examination was normal in both eyes, and the vitreous was clear. Optical coherence tomography (OCT) of the macular and optic nerve in both eyes was normal. The endothelial cell count in both eyes exceeded 2000 cells/mm2. He had been told by a previous ophthalmologist to have bilateral cataracts and was now keen for surgery starting with the right eye. He works as a manager in a large company and is an avid golf player. He is emmetropic and wears spectacles for near work. Optical biometry using predicated posterior corneal astigmatism did not recommend a toric intraocular lens (IOL). He had done his internet research on IOL options and requested trifocal IOLs. What is your surgical plan for right cataract removal? Explain which IOL you would choose to implant.

E3 ubiquitin protein ligase, E6-associated protein (E6-AP) regulates PI3K-Akt signaling and prostate cell growth.

This study elucidates the role of E6-associated protein, E6-AP (a dual function steroid hormone receptor coactivator and ubiquitin-protein ligase) in the regulation of PI3K-Akt signaling pathway, prostate gland growth and proliferation. Here, we report the generation of transgenic mice and prostate cancer cell line, LNCaP cells that overexpress E6-AP protein. Using these models we show that the levels of total Akt and phosphorylated Akt (active Akt) are increased in E6-AP overexpressing prostate gland and LNCaP cells suggesting that E6-AP regulates the PI3K-Akt signaling pathway. The prostate glands in our transgenic mice are ~20% larger and produce preneoplastic lesions at the age of 18 months. Our data also suggest that E6-AP modulates PI3K-Akt signaling pathway by both androgen-independent and -dependent mechanisms. In the androgen-independent mechanism, E6-AP modulates PI3K-Akt signaling by regulating the protein levels of RhoA, a small GTPase, which is a negative regulator of the Akt signaling pathway. Further, we show that E6-AP, a known coactivator of AR, amplifies the androgen-dependent activation of PI3K-Akt signaling pathway. In addition, we show that stable overexpression of E6-AP in prostate cancer cells results in increased cell size and proliferation. Overall our data suggests that E6-AP regulates both the positive and negative modulators of the PI3K-Akt pathway in prostate cells which results in increased prostate cell growth, proliferation and decreased apoptosis.This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

Bicyclic core estrogens as full antagonists: synthesis, biological evaluation and structure-activity relationships of estrogen receptor ligands based on bridged oxabicyclic core arylsulfonamides.

Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester (termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ERα and ERß. Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its partial antagonist character by stabilizing a novel conformation of the ER that involves a significant distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or tertiary sulfonamides (-SO(2)NR-), isoelectronic and potentially isostructural molecular replacements. An array of 16 OBHS sulfonamide analogues were prepared through a Diels-Alder reaction of a 3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity. In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower intrinsic transcriptional activity on ERα than the selective estrogen receptor modulator (SERM) hydroxytamoxifen or OBHS, and they were inverse agonists on ERß. Thus, the behaviour of these OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant (ICI 182 780), and their greater antagonist biocharacter appears to arise from an accentuated distortion of helix-11.

Balloon cell naevus of the conjunctiva: clinicopathological features and management.

A 7-year-old Caucasian girl presented with a pigmented lesion on the left bulbar conjunctiva that increased in size from 1 mm to 4 mm over a 12-month period. She underwent excision biopsy and reconstruction of the ocular surface with amniotic membrane graft. Histopathology showed the naevus was composed of somewhat swollen naevus cells with clear cytoplasm and central nucleus. These vacuolated naevus cells were approximately 40 µm in diameter. Over 90% of the cells in the naevus were composed of these swollen cells. Immunohistochemical staining was positive for S100 and Melan-A. This case illustrates that balloon cells may be observed in conjunctival naevi at a previously unreported pre-pubescent age. Awareness of ballon cell naevus is important to avoid clinical and histological pitfalls in diagnosis.

Whole-mount Immunohistochemistry to Visualize Mouse Embryonic Dermal Lymphatic Vasculature.

Lymphatic vessels are abundant in the skin where they regulate interstitial fluid uptake and immune surveillance. Defects in dermal lymphatic vessels, such as fewer vessels and abnormal lymphatic vessel coverage with mural cells, are frequently associated with lymphedema and other lymphatic disorders. Whole-mount immunohistochemistry allows the visualization of dermal lymphatic vessels and identifies morphogenetic defects. Most dermal lymphatic vessels start growing during embryogenesis from lymph sacs that are located close to the axilla towards the dorsal and ventral midlines. Here, we present an approach that we have developed to permeabilize, immunolabel, clear, and visualize the lymphatic vessels. These simple and inexpensive techniques reproducibly generate images of dermal lymphatic vessels with great clarity.

Long-term performance of a diffractive-refractive trifocal IOL with centralized diffractive rings: 5-year prospective clinical trial.

PURPOSE: To report the 5-year visual, refractive, and patient-reported outcomes following implantation of a trifocal intraocular lens (IOL) during cataract surgery. SETTING: Csolnoky Ferenc University Hospital, Veszprém, Hungary. DESIGN: Prospective, longitudinal, single-center, interventional study. METHODS: 100 eyes of 50 patients underwent bilateral implantation of a trifocal IOL during cataract surgery. Preoperative corrected distance (CDVA) and postoperative uncorrected distance visual acuity (UDVA), CDVA, uncorrected (UIVA) and corrected (CIVA) intermediate and uncorrected (UNVA) and corrected (CNVA) near visual acuity were collected. All subjects were seen at day 1, 1 month, 3 months, 6 months, 12 months, and 24 months, and at year 5. Contrast sensitivity, slitlamp photography, and quality of vision questionnaire were performed at months 3, 6, 12, and 24 and at year 5. Of these 50 patients, 41 completed their 5-year follow-up. RESULTS: At year 5, 74 eyes of 37 patients were analyzed. The mean postoperative UDVA was 0.02 ± 0.10 (logMAR). The mean CDVA was -0.04 ± 0.07. The mean UIVA was 0.04 ± 0.09. The mean CIVA was 0.00 ± 0.08. The mean UNVA was 0.09 ± 0.09. The mean CNVA was 0.05 ± 0.07. Mesopic and photopic contrast sensitivity values were in the upper third range of the age-matched normal values. CONCLUSIONS: 5-year prospective study data showed that bilateral implantation of a diffractive-refractive trifocal IOL with centralized diffractive rings provided good functional vision at all distances. There was high level of spectacle independence and patient satisfaction with minimal levels of dysphotopsia.